User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/12

ADA Kinetic Assay

• The procedure was taken from Dhea Patel's notebook entry.
• The assay runs were continued from last week's, 02/05/2013, calculated values.
• The objective of performing the assay is to obtain the rate of the enzymatic reaction of adenosine into inosine by ADA.
• Baseline 200-400nm using pH 7.4, 0.05M phosphate buffer
• Temperature control: 25°C
• Phosphate buffer in reference cell
• Ran kinetics on 250uM solution (20uL ADA, 600uL 1250uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
  • absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.
• Ran kinetics on 2.56uM solution (20uL ADA, 600uL 12.8uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
  • dAbs was obtained and plotted vs. time in excel
• Ran kinetics on 6.4uM solution (20uL ADA, 600uL 32uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
  • dAbs was obtained and plotted vs. time in excel
• Ran kinetics on 16uM solution (20uL ADA, 600uL 80uM Adenosine, and 2.38mL pH7.4, 0.005M phosphate buffer) for 5 minutes at 265nm.
  • dAbs was obtained and plotted vs. time in excel
• The following table describes the volumes and concentrations of the components added to the cuvette.

• Note: Since the 250 μM adenosine assay exhibited a high absorbance, this concentration was replaced with two other adenosine concentrations, 150 and 200 μM.
• The stock solution for 150 μM adenosine was made by diluting 150 μL of 5 mM adenosine into 850 ng/μL of 0.05 M phosphate buffer. The 200 μM adenosine was made by diluting 200 μL of 5 mM adenosine into 800 μL of 0.05 M phosphate buffer.